Interest in proteomic analysis of human serum has been greatly elevated during the past several years. Proteomic analysis of human serum represents an extreme challenge due to the dynamic range of the proteins of interest. In serum, the quantities of proteins and peptides of interest range from those considered “high abundance”, present at 2-50% by mass of total protein, to those present at 10−12 or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. One way to address the complexity of these samples is the application of multidimensional separation techniques.
At present, two major approaches are being used as prefractionation techniques: chromatographic and electrophoretic. Liquid chromatography equipment and methods, such as high-performance liquid chromatography (HPLC), can be used in a variety of applications, such as, for example, in chemical analysis or diagnostics, to separate, isolate, and identify chemical compounds in mixtures. A significant problem encountered in liquid chromatographic methods is the lack of methods to reliably separate, detect, or distinguish low abundance protein constituents from high abundance protein constituents in a sample of interest. Consequently, lower abundance proteins, for example, of significant biological interest, can be incompletely detected or can go undetected or overlooked. Additionally, recovery of proteins from currently used HPLC columns is often unsatisfactory for proteomic analysis since lower abundance proteins may be present at concentrations that are at or below the detection limits of conventional instrumentation used in proteomic analysis (e.g., such as mass spectrometers).